U.S. patent application number 10/686355 was filed with the patent office on 2004-06-03 for antibodies active against a fusion polypeptide comprising a histidine portion.
This patent application is currently assigned to Deutsches Krebsforschungszentrum Stiftung des Offentlichen Rechts. Invention is credited to Frey, Manfred, Schwinn, Susanne, Tessmer, Claudia, Velhagen, Iris, Zentgraf, Hanswalter.
Application Number | 20040105854 10/686355 |
Document ID | / |
Family ID | 7755368 |
Filed Date | 2004-06-03 |
United States Patent
Application |
20040105854 |
Kind Code |
A1 |
Zentgraf, Hanswalter ; et
al. |
June 3, 2004 |
Antibodies active against a fusion polypeptide comprising a
histidine portion
Abstract
The present invention relates to an antibody active against a
fusion polypeptide comprising a histidine portion, a process for
the preparation thereof and its use.
Inventors: |
Zentgraf, Hanswalter;
(Heidelberg, DE) ; Tessmer, Claudia; (Schwarzach,
DE) ; Velhagen, Iris; (Schwetzingen, DE) ;
Schwinn, Susanne; (Hockenheim, DE) ; Frey,
Manfred; (Mannheim, DE) |
Correspondence
Address: |
DORSEY & WHITNEY LLP
INTELLECTUAL PROPERTY DEPARTMENT
4 EMBARCADERO CENTER
SUITE 3400
SAN FRANCISCO
CA
94111
US
|
Assignee: |
Deutsches Krebsforschungszentrum
Stiftung des Offentlichen Rechts
|
Family ID: |
7755368 |
Appl. No.: |
10/686355 |
Filed: |
October 14, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10686355 |
Oct 14, 2003 |
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08913139 |
Feb 9, 1998 |
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08913139 |
Feb 9, 1998 |
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PCT/DE96/00369 |
Mar 1, 1996 |
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Current U.S.
Class: |
424/130.1 ;
435/7.1; 435/7.92; 435/70.21; 530/388.1 |
Current CPC
Class: |
C07K 16/32 20130101;
C07K 16/1054 20130101 |
Class at
Publication: |
424/130.1 ;
435/007.1; 530/388.1; 435/070.21; 435/007.92 |
International
Class: |
G01N 033/53; C12P
021/04; A61K 039/395; C07K 016/18 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 1, 1995 |
DE |
195 07 166.2 |
Claims
1. An antibody against a fusion polypeptide comprising a histidine
portion.
2. The antibody according to claim 1, characterized in that it is
polyclonal.
3. The antibody according to claim 1, characterized in that it is
monoclonal.
4. The antibody according to claim 3, characterized in that it is
deposited under ACC 2207 with DSM [German-type culture collection
for microorganisms].
5. A process for the preparation of an antibody according to any
one of claims 1 to 4, characterized in that an animal is immunized
with a histidine fusion polypeptide and (a) polyclonal antibodies
are obtained from the serum of the animal, or (b) monoclonal
antibodies are obtained after the fusion of animals spleen cells
with myeloma cells.
6. The process according to claim 5, characterized in that a
mixture of histidine fusion polypeptides is used for
immunization.
7. Use of an antibody according to any one of claims 1 to 4 in a
detection method for a fusion polypeptide comprising a histidine
portion.
8. Use according to claim 7, wherein the detection method is a
Western blot, an ELISA, an immunofluorescence or an
immunoprecipitation.
Description
[0001] The present invention relates to antibodies which are active
against a fusion polypeptide comprising a histidine portion, a
process for the preparation thereof and their use.
[0002] It is known to express a polypeptide in the form of a
histidine fusion polypeptide. In such a polypeptide, a histidine
portion of e.g. 6-18 successive histidine residues is fused to the
C or N terminus of the polypeptide. Hence it is possible to isolate
the histidine fusion polypeptide by means of a nickel-chelate
chromatographic column from the supernatant or cell lysate of the
cell expressing it.
[0003] However, the above column is expensive. Furthermore, its use
costs a lot of time. Therefore, it is not suited for the rapid
detection of the expression of a histidine fusion polypeptide. But
such a detection is necessary, particularly when it shall be used
for screening many cells.
[0004] Thus, it is the object of the present invention to provide
means by which the expression of a histidine fusion polypeptide can
be detected rapidly.
[0005] According to the invention this is achieved by an antibody
which is directed against a fusion. polypeptide comprising a
histidine portion.
[0006] Such an antibody may be a polyclonal or monoclonal antibody,
a monoclonal antibody being preferred. The antibody may be obtained
from any animal or human being, rabbits being preferred for a
polyclonal antibody and mice being preferred for a monoclonal
antibody.
[0007] In addition, the antibody may be synthetic, portions which
are not necessary for the above-mentioned identification optionally
lacking fully or partially therefrom and these portions being
replaced by others which give the antibody further favorable
properties, respectively.
[0008] The expression "fusion polypeptide comprising a histidine
portion" comprises a polypeptide (peptide) of any kind and length
which has a histidine portion. Such a polypeptide may be expressed
by any cells, e.g. bacteria, yeasts, cells of insects, plants and
animals, as well as organisms, e.g. transgenic animals. An above
histidine portion may comprise e.g. 6-18, preferably 6, successive
histidine residues and be fused to the N and/or C terminus of the
polypeptide.
[0009] A preferred antibody of the present invention, namely a
monoclonal mouse antibody having the above identification, was
deposited under No. ACC 2207 with the DSM [German-type collection
of microorganisms] on Feb. 15, 1995.
[0010] Antibodies according to the invention can be prepared
according to conventional methods. If polyclonal antibodies and
monoclonal antibodies, respectively, are to be prepared, it will be
favorable to immunize animals, particularly rabbits for the former
antibodies and mice for the latter antibodies, with an above
histidine fusion polypeptide e.g. His p53 (cf. German patent
application P 42 32 823.3) or His hdm2 (cf. German patent
application P 43 39 553.3), preferably a mixture thereof. The
animals can be further boostered with the same histidine fusion
polypeptide or polypeptides. Other histidine fusion polypeptides or
a combination of these and the preceding histidine fusion
polypeptide or polypeptides may also be used for boostering. The
polyclonal antibodies may then be obtained from the serum of the
animals. Spleen cells of the animals are fused with myeloma cells
for the monoclonal antibodies.
[0011] For the preparation of synthetic antibodies, e.g. the
above-obtained monoclonal antibodies may be used as a basis. For
this purpose, it is the obvious thing to analyze the
antigen-binding region of the monoclonal antibodies and identify
the portions which are necessary and not necessary for the above
identification. The necessary portions may then be modified and the
non-necessary portions can be fully or partially eliminated and
replaced by portions giving the antibodies further favorable
properties, respectively. Also, portions can be modified,
eliminated or replaced beyond the binding regions of the
antibodies. A person skilled in the art knows that particularly the
DNA recombination technology is suitable for the above measures. He
is perfectly familiar therewith.
[0012] Antibodies according to the invention distinguish themselves
in that they recognize any fusion polypeptides comprising a
histidine portion. Therefore, the antibodies are suitable for the
rapid detection of the expression of such fusion polypeptides. This
may be carried out in any detection methods, particularly in a
Western blot, an ELISA, an immunoprecipitation or an
immunofluorescence. For this purpose, the antibodies according to
the invention may be labeled, if appropriate, or used in
combination with labeled antibodies directed thereagainst.
[0013] The present invention is explained by the below
examples.
EXAMPLE 1
Preparation of Monoclonal Antibodies
[0014] Mice were used for immunization. His hdm2 (amino acid
1-284), His hdm2 (amino acid 58-491) and His p53 (amino acid
66-393) (cf. above) were used as antigens. They were dissolved in a
buffer comprising 8 M urea, 100 mM, NaH.sub.2PO.sub.4, 10 mM
Tris-HCl.
[0015] Immunization and Booster Pattern
1 Day 1: 50 .mu.l (= 10 .mu.g) His hdm2 (amino acid 1-284) 50 .mu.l
(= 10 .mu.g) His hdm2 (amino acid 58-491) 50 .mu.l PBS
(phosphate-buffered saline) 150 .mu.l Freund's adjuvant complete
300 .mu.l mix 200 .mu.l of the mix were injected into a mouse Day
30: 50 .mu.l (= 10 .mu.g) His hdm2 (amino acid 1-284) 50 .mu.l (=
10 .mu.g) His hdm2 (amino acid 58-491) 20 .mu.l PBS 120 .mu.l
Freund's adjuvant incomplete 240 .mu.l mix 200 .mu.l of the mix
were injected into the above mouse. Day 60: 50 .mu.l (= 10 .mu.g)
His hdm2 (amino acid 1-284) 50 .mu.l (= 10 .mu.g) His hdm2 (amino
acid 58-491) 85 .mu.l PBS 115 .mu.l Freund's adjuvant incomplete
300 .mu.l mix 200 .mu.l of the mix were injected into the above
mouse. Day 90: 50 .mu.l (= 10 .mu.g) His hdm2 (amino acid 1-284) 50
.mu.l (= 10 .mu.g) His hdm2 (amino acid 58-491) 200 .mu.l PBS 300
.mu.l mix 200 .mu.l of the mix were injected into the above mouse.
Day 180: 150 .mu.l (= 20 .mu.g) His p53 (amino acid 66-393) 150
.mu.l Freund's adjuvant complete 300 .mu.l mix 200 .mu.l of the mix
were injected into the above mouse. Day 230: 75 .mu.l (= 10 .mu.g)
His p53 (amino acid 66-393) 25 .mu.l (= 5 .mu.g) His hdm2 (amino
acid 1-284) 25 .mu.l (= 5 .mu.g) His hdm2 (amino acid 58-491) 125
.mu.l Freund's adjuvant incomplete 250 .mu.l mix 200 .mu.l of the
mix were injected into the above mouse. Day 260: 75 .mu.l (= 10
.mu.g) His p53 (amino acid 66-393) 25 .mu.l (= 5 .mu.g) His hdm2
(amino acid 1-284) 25 .mu.l (= 5 .mu.g) His hdm2 (amino acid
58-491) 125 .mu.l PBS 250 .mu.l mix 200 ml of the mix were injected
into the above mouse.
[0016] The mouse was killed on day 262. Spleen cells were removed
therefrom and fused with myeloma cells. Monoclonal antibodies were
obtained. One of them was deposited under ACC 2207 with DSM on Feb.
15, 1995.
[0017] Preparation of Polyclonal Antibodies
[0018] Rabbits were used for immunization. The antigens of Example
1 were employed. The immunization and booster pattern was identical
with that of Example 1 up to day 90 inclusive.
2 Day 92: 5 ml of blood were removed from the rabbit's auricular
vein and tested for antibody activity in an ELISA and Western blot,
respectively. Day 93: Following a positive test on day 92, the
animals were killed and the antibodies were obtained from the
serum.
EXAMPLE 3
Detection of Histidine Fusion Polypeptides by Antibodies According
to the Invention
[0019] (a) Western Blot
[0020] Histidine fusion polypeptides, namely His hdm2 (amino acid
1-284), His hdm2 (amino acid 58-491) and His p53 (amino acid
66-393) of Example 1, as well as the polypeptides hdm2 (amino acid
1-284), WAF 1 (=wild type-activating factor) and t16
(=cell-regulating protein) as control were subjected to a
polyacrylamide gel eletrophoresis. The gel was transferred
overnight to a nitrocellulose membrane. It was then incubated with
the above antibody ACC 2207 diluted in a ratio of 1:10 and 1:50,
respectively, at 37.degree. C. for 1 hour. After several wash steps
using PBS (0.05% Tween 20), a purchasable alkaline
phosphatase-coupled goat-anti-mouse antibody (dilution according to
the manufacturer's indication) was added. A 30-minute incubation at
37.degree. C. was followed by several wash steps using PBS and
thereafter the alkaline phosphatase detection reaction with
alkaline phosphatase including developing solution (36 .mu.M
5'-bromo-4-chloro-3-indolylphosphate, 400 .mu.M nitroblue
tetrazolium, 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl.sub.2)
at room temperature until bands were visible.
[0021] It showed that the antibody ACC 2207 according to the
invention recognizes specifically histidine fusion polypeptides but
not polypeptides without histidine portion.
[0022] (b) ELISA
[0023] A 96-well plate was provided per well with 100 .mu.l each,
which included 20 ng and 8 ng, respectively, of the histidine
fusion polypeptides and the controls of (a), respectively. After
incubation at 4.degree. C. overnight, 3 short wash steps using PBS
followed. Thereafter, the free binding sites of the polymeric
carrier were blocked by one-hour incubation using 1% BSA in PBS at
37.degree. C. The antibody ACC 2207 according to the invention
which was diluted in a ratio of 1:10 and 1:50, respectively, was
incubated on the plate at 37.degree. C. for 1 hour. After 8 wash
steps using PBS, the peroxidase-coupled goat anti-mouse antibody of
(a) was added. A 30-minute incubation at 37.degree. C. was followed
by 8 wash steps and thereafter the peroxidase detection reaction
with developing solution (50 mM sodium acetate, 0.4 mM
3,3',5,5'-tetramethylbenzidine dihydrochloride, 4.4 mM
H.sub.2O.sub.2) at room temperature until bands were visible.
[0024] It showed that the antibody ACC 2207 according to the
invention recognizes specifically histidine fusion polypeptides but
not a polypeptide without histidine portion.
* * * * *